Fig. 5: Validation of the protrusion of SD domains out of the Xist territory and their closer proximity in SmcHD1-mutant NSCs by FISH.
From: Replication dynamics identifies the folding principles of the inactive X chromosome
a, SD, SI and CL domain BACs were used to examine their subnuclear localization relative to the Xist cloud. b, Representative images of RNA- and DNA-FISH using Xist RNA and SD, SI or CL BAC probes in JB4/EI7HZ2 NSCs. We categorized the DNA-FISH signal localization relative to the Xist cloud into four groups (Methods). Scale bars, 5 μm. c,e, Percentages of DNA-FISH signal localization relative to the Xist cloud in NSCs: set 1 (c) and set 2 (e). d,f, Distance between the DNA-FISH signal and the Xist cloud centroid in NSCs normalized with Xist Feret diameter in the same nucleus: set 1 (d) and set 2 (f). g, Three SD BACs and their interaction frequencies as observed by 4C-seq (Fig. 3b and Supplementary Fig. 6). Red lines, viewpoints; blue lines, Dxz4; gray bars beneath each plot, significant far-cis contacts; colored bars, RT classes. h, Representative images of RNA- and DNA-FISH using Xist RNA and SD BAC probes in JB4/EI7HZ2 NSCs. The SD–SD localization patterns were categorized into three groups. Scale bars, 5 μm. i, Percentages of SD–SD localization patterns in WT and SmcHD1-mutant NSCs. j,k, Normalized distances between two SD probes were plotted as cumulative plots: comparing WT versus SmcHD1-mutant NSCs (j) and comparing the SD protrusion group (k). N, the total number of cells analyzed from two to three independent experiments. P values in c, e and i were obtained from a chi-square test for all groups and a Fisher’s exact test for the protruded group. P values in d and f were obtained from one-sided Wilcoxon signed-rank test, with data that used a paired test indicated. P values in j and k were obtained from a two-sided Kolmogorov–Smirnov test.
